A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.